Bottom Line:
We used in vivo time-lapse quantitative microscopy to show that clathrin, dynamin, and the ADP-ribosylation factor 6, three components of the endocytic machinery, accumulate around wounds in Drosophila melanogaster embryos in a process that requires calcium signaling and actomyosin contractility.Blocking endocytosis with pharmacological or genetic approaches disrupted wound repair.Reducing E-cadherin levels in embryos in which endocytosis was blocked rescued actin localization to the wound margin.

Mentions:
What signal polarizes the endocytic machinery around embryonic wounds? Transient, large calcium stimuli promote endocytosis at nerve terminals in rat brainstems (Wu and Wu, 2014). Recent studies showed that wounding causes a transient calcium wave that decreases in amplitude as it moves away from the wound (Antunes et al., 2013; Razzell et al., 2013). Therefore, a calcium signal may promote polarized endocytosis in the cells adjacent to the wound. To investigate whether a calcium signal was implicated in the recruitment of the endocytic machinery to the wound margin, we injected embryos with 50 mM BAPTA, a cell-impermeable calcium chelator. In embryos expressing GCaMP3, a fluorescent calcium biosensor (Nakai et al., 2001; Tian et al., 2009), BAPTA injection immediately before wounding completely attenuated the calcium signal caused by wounding (Video 7). When we compared the localization of the endocytic machinery during wound repair in water or BAPTA-injected embryos, we found that in water-injected embryos dynamin:GFP accumulated at the wound margin to a maximum level of 2.5 ± 0.4-fold (Fig. 5, A, C, and D; and Video 8). In contrast, in BAPTA-treated embryos, mean dynamin fluorescence at the wound margin only increased by 1.2 ± 0.1-fold, a value 52% lower than in controls (P = 1.6 × 10−2; Fig. 5, B–D; and Video 8). Similarly, GFP:clc fluorescence at the wound margin in BAPTA-injected embryos displayed a maximum increase of 1.4 ± 0.1-fold, significantly lower than the 1.9 ± 0.2-fold enrichment in control embryos (P = 1.7 × 10−2; Fig. S4, A–D). Together, these results indicate that the polarized accumulation of the endocytic machinery to the wound margin is mediated by the release of calcium in the epidermis upon wounding, suggesting that calcium signaling may be important for cytoskeletal and junctional remodeling in embryonic wound repair.

Mentions:
What signal polarizes the endocytic machinery around embryonic wounds? Transient, large calcium stimuli promote endocytosis at nerve terminals in rat brainstems (Wu and Wu, 2014). Recent studies showed that wounding causes a transient calcium wave that decreases in amplitude as it moves away from the wound (Antunes et al., 2013; Razzell et al., 2013). Therefore, a calcium signal may promote polarized endocytosis in the cells adjacent to the wound. To investigate whether a calcium signal was implicated in the recruitment of the endocytic machinery to the wound margin, we injected embryos with 50 mM BAPTA, a cell-impermeable calcium chelator. In embryos expressing GCaMP3, a fluorescent calcium biosensor (Nakai et al., 2001; Tian et al., 2009), BAPTA injection immediately before wounding completely attenuated the calcium signal caused by wounding (Video 7). When we compared the localization of the endocytic machinery during wound repair in water or BAPTA-injected embryos, we found that in water-injected embryos dynamin:GFP accumulated at the wound margin to a maximum level of 2.5 ± 0.4-fold (Fig. 5, A, C, and D; and Video 8). In contrast, in BAPTA-treated embryos, mean dynamin fluorescence at the wound margin only increased by 1.2 ± 0.1-fold, a value 52% lower than in controls (P = 1.6 × 10−2; Fig. 5, B–D; and Video 8). Similarly, GFP:clc fluorescence at the wound margin in BAPTA-injected embryos displayed a maximum increase of 1.4 ± 0.1-fold, significantly lower than the 1.9 ± 0.2-fold enrichment in control embryos (P = 1.7 × 10−2; Fig. S4, A–D). Together, these results indicate that the polarized accumulation of the endocytic machinery to the wound margin is mediated by the release of calcium in the epidermis upon wounding, suggesting that calcium signaling may be important for cytoskeletal and junctional remodeling in embryonic wound repair.

Bottom Line:
We used in vivo time-lapse quantitative microscopy to show that clathrin, dynamin, and the ADP-ribosylation factor 6, three components of the endocytic machinery, accumulate around wounds in Drosophila melanogaster embryos in a process that requires calcium signaling and actomyosin contractility.Blocking endocytosis with pharmacological or genetic approaches disrupted wound repair.Reducing E-cadherin levels in embryos in which endocytosis was blocked rescued actin localization to the wound margin.